Revision as of 15:48, 24 July 2009

Protocol for transforming (E.coli)

Take 2 and 5 µl of the plasmid from the distribution plates and add it to 2 different tubes with 50 µl competent cells in each. As controls use 1 tube with competent cells only (control of the cells) and 1 tube with puc plasmid which is supercoiled and known to work (control of our plasmid).
Store on ice for 40 min.
Keep at 42º C for 2 min.
Keep on ice for 5 min.
Add 1 ml LB to each tube.
The tubes are stored at 37 º C for 2 hours while being shaken.
Centrifuge the tubes at 3500 rounds pr. minute for 5 min.
Suck up and throw out 850 µl and resuspend the remaining by pipetting up and down.
Spread out 75 µl on 2 LA (LB + agarose) plates with antibiotics (100 µg/ml ampicillin). The competent cells of the controle are spread out on 1 LA plate with antibiotics and 1 without, since they are not resistant.
Store at 37 º C over night.

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-# Take 2 and 5 µl of the plasmid from the distribution plates and add it to 2 different tubes with 50 µl competent cells in each. As controls use 1 tube with competent cells only (controle of the cells) and 1 tube with puc plasmid which is supercoiled and known to work (controle of our plasmid).
+# Take 2 and 5 µl of the plasmid from the distribution plates and add it to 2 different tubes with 50 µl competent cells in each. As controls use 1 tube with competent cells only (control of the cells) and 1 tube with puc plasmid which is supercoiled and known to work (control of our plasmid).
 # Store on ice for 40 min.
 # Keep at 42º C for 2 min.